CN116200500A - S1pr1作为白血病干细胞分子标志物的应用 - Google Patents
S1pr1作为白血病干细胞分子标志物的应用 Download PDFInfo
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Abstract
本发明涉及白血病诊断或预后评估领域，具体来说涉及一种急性髓系白血病干细胞分子标志S1PR1，其可应用于相关疾病的诊断和治疗，还涉及相关诊断试剂盒和治疗产品。
Description
技术领域
本发明属于生物技术领域，尤其涉及S1PR1作为一种具有调控功能的AML白血病干细胞表面标志物。
背景技术
白血病(leukemia)是一类造血细胞异常增殖的恶性疾病，在我国被列为十大高发恶性肿瘤之一，在儿童和35岁以下成人中居第一位。其中，急性白血病属造血干细胞恶性克隆性疾病，主要是由于造血干细胞、祖细胞发生基因表达谱的改变所引发的恶性增生，临床表现为患者正常造血功能受到抑制，大量白血病细胞在骨髓中增殖聚积并浸润髓外器官和组织。患者容易出现贫血、出血、感染及各器官浸润等症状。
急性髓系白血病(acute myeloid leukemia,AML)是一种髓系造血干/祖细胞恶性疾病，属于最常见但也最为致命的白血病类型之一，其中难治及复发AML的诊疗尤其面临着极大的挑战。美国癌症协会(American Cancer Society,ACS)的统计数据表明，2020年美国共有约60,530例新发白血病病例和23,100例白血病死亡病例，其中AML新发病例约19,940例，并约有11,180人死于AML。白血病被认为是正常造血干祖细胞发生基因表达谱改变的结果，具有自我更新、分化和无限增殖能力的白血病干细胞(leukemia stem cells,LSCs)的存在被认为是白血病治疗后耐药和复发的根源。白血病干细胞是一群具有自我更新能力和无限的替代潜力，并能产生异质性白血病细胞群体的白血病细胞，被发现与AML的化疗抵抗和复发有关。经研究，一些细胞信号通路在AML的存活、增殖及自我更新中起重要作用，并且在LSC中被异常激活或抑制，包括NF-κB、Wnt/β-catenin、Hedgehog、Notch、EGFR、JAK/STAT、PI3K/AKT/mTOR、TGF/SMAD和PPAR通路等。因此，这些信号转导途径成为了AML治疗的研究热点，LSC相关信号通路抑制剂/激活剂被认为是一种潜在的新型抗AML疗法，通过消除LSC以指导急性髓系白血病的研究及临床治疗。
经过多年的临床实践，一般而言，年龄<60岁的患者长期生存率为35％～45％，年龄≥60岁的患者长期生存率仅为10％～15％。随着近年来各种治疗方法的发展，白血病化疗的完全缓解率(complete remission，CR)有所提高，但仍有60％～70％的患者治疗后耐药或复发，白血病复发是提高AML疗效的主要瓶颈。
科学家通过分离白血病细胞并打入小鼠体内后发现只有少量细胞可以诱发白血病首次阐明了肿瘤干细胞(cancer stem cell,CSC)的存在。随后科学家又发现了CD34+CD38-可以作为LSC的表面标志物。此后随着学者对LSC研究的不断深入，逐步发现了更多的AML中LSC的标志物，例如HMGN1,JAM3,FOXM1,GPR56,CD200和CD93等。其中，很多LSC的表面标志物被验证具有调控LSC自我更新及重建白血病等生物学特性的作用。细胞遗传学分析为AML提供了不同亚型的预后信息，而近来已越来越多地发现与预后相关的各种分子标志。这些发现使得对不同预后的AML的分析进入分子水平。
在临床实践中，通常可使用分子标志，如白血病融合基因RUNX1-RUNX1T1、CBFβ-MYH11和PML-RARα以及NPM1突变预测AML复发。获得CR的AML患者体内可检测到DNMT3A、ASXL1和TET2等前白血病克隆相关突变，由于这些突变也可见于健康人群，且发生率随年龄增大而增加，也被称为年龄相关的克隆造血或意义未名的克隆性造血，因此，这些突变的复发预测意义仍待证实。其他分子标志，例如FLT3-ITD、FLT3-TKD、NRAS、KRAS、IDH1和IDH2等部分基因突变在AML患者复发时存在不稳定现象(如初诊阳性突变，复发时可阴性，或初诊阴性而复发时新出现阳性)，会明显增高假阳性和假阴性率。可见，仍然需要发现和研究其他类型的具有诊断以及治疗作用的白血病相关标记物。
白血病干细胞的自我更新和耐药的特性正是由异常表达的基因所维持，如果能找到特定基因作为精准诊断、预后评价和治疗的靶点，对于白血病患者疾病进展监控、实现长期生存具有重要意义。科学评估急性白血病的诊断、治疗及预后相关的辅助指标，对于选择有效的化疗方案或进行骨髓移植有着重要的临床意义。目前，实验室诊断急性白血病的指标主要包括骨髓涂片细胞形态学检查、流式细胞免疫分型、外周血全血细胞计数以及遗传学检查等，随着检测技术的发展，越来越倾向于使用对患者微创、自动检测、有标准规范且易于推广的快速检测指标。
发明内容
本发明的发明人在多年的研究中发现S1PR1对白血病干细胞的自我更新能力具有调控作用。本发明阐明了S1PR1对于白血病干细胞的调控作用。本发明还提供一种新型的白血病干细胞标志物，所述标记物可用于相关疾病的复发率评估，疾病风险评估，预后评估，分层诊断，检测或者辅助检测病程进展情况，以及相关疾病的治疗或相关药物筛选等，还可利用该标记物制备白血病模型小鼠以用于科研。
本发明人在多年的研究中，发现鞘氨醇-1-磷酸受体1(sphingosine-1-phosphatereceptor 1,S1PR1)对白血病干细胞的自我更新及重建白血病的能力具有调控作用，并验证了S1PR1对于白血病干细胞的调控作用。鞘氨醇-1-磷酸(sphingosine-1-phosphate，S1P)是具有生物活性的鞘脂代谢产物，通过与特异性的G蛋白偶联受体结合，广泛参与调控多种生物学功能。目前已发现的S1P受体家族有5个成员，分别是S1PR1、S1PR2、S1PR3、S1PR4和S1PR5。
有研究表明，S1PR1在人肺组织中，抑制S1PR1基因的表达会导致肺毛细血管漏出增加，活化的S1PR1能调控RacGTP酶依赖的皮层肌动蛋白重排，增强内皮细胞的屏障作用。S1PR1能稳定VE-Cadherin的定位并抑制VEGF诱导的VEGFR2激活，从而抑制血管的出芽并增强内皮细胞的连接性。2019年，有研究者(褚福营等，LDB2在肺癌组织中的表达及与S1PR1的相关性分析，《临床检验杂志》)研究了S1PR1基因在肺癌组织中的表达，并分析了其与LIM域结合蛋白2(LDB2)基因的相关性，结果发现，S1PR1基因在肺癌组织中表达量为0.710(0.337～1.523)，在癌旁组织中表达量为1.582(0.913～3.533)，其表达水平显著降低且与LDB2基因存在强相关性，这表明LDB2与S1PR1基因在肺癌血管生成的生物学过程中可能具有一定的协同作用，但两者具体的调节机制还需要进一步验证。该研究阐释了S1PR1在肺癌中的表达情况，然而肺癌属于实体肿瘤，与白血病发病机制、治疗机理完全不同，这两种疾病的分子标记物也千差万别。
本发明在第一方面提供了白血病干细胞分子标志物S1PR1在制备用于白血病诊断或治疗预后监测的试剂或试剂盒中的应用。还提供了S1PR1作为白血病干细胞分子标志物的应用。尤其是，S1PR1作为白血病干细胞分子标志物的应用，所述标记物能够调控白血病干细胞的自我更新能力。
进一步的，所述白血病为急性白血病，优选为急性髓系白血病。
进一步的，所述诊断疾病进展或评价预后是包括：所述1)S1PR1在急性髓系白血病患者骨髓血单个核细胞较正常供者来源的骨髓血单个核细胞高表达，；且2)S1PR1在急性髓系白血病干细胞较非干性白血病细胞高表达，；特别3)S1PR1在在难治和复发后的标本中异常高表达，；或4)S1PR1在自我更新及重建白血病能力强的髓系白血病细胞系和耐药细胞系中高表达。
本发明在第二方面提供了S1PR1在制备用于治疗或预防白血病的药物中的应用。
进一步的，所述白血病为急性白血病，优选为急性髓系白血病。
进一步的，所述药物为S1PR1的抑制剂。
优选的，所述S1PR1的抑制剂是抑制ZNF683蛋白质活性或蛋白质水平的抑制剂、或者抑制S1PR1的mRNA水平的抑制剂，其抑制活性是可逆的或不可逆的。
更优选的，所述抑制S1PR1蛋白质活性或蛋白质水平的抑制剂包括S1PR1的抗体、抑制S1PR1蛋白质活性或蛋白质水平的蛋白质、多肽、酶、天然化合物、合成化合物、有机物、无机物；所述抑制S1PR1蛋白质活性或蛋白质水平的抑制剂是指可以结合S1PR1但在结合时不产生生物应答的物质，或者所述抑制剂可以阻断、抑制或减弱由激动剂介导的应答；所述抑制S1PR1蛋白质活性或蛋白质水平是指敲除S1PR1。
优选的，所述抑制S1PR1的mRNA水平的抑制剂是其反义核酸序列、siRNA、miRNA、shRNA、dsRNA，或者其它能够抑制S1PR1的mRNA水平的蛋白质、多肽、酶、化合物。
本发明在第三方面提供了一种制备白血病模型小鼠的方法，其特征在于，所述方法包括以下步骤：1)分选出AML患者骨髓血中CD34+S1PR1+细胞，2)小鼠尾静脉注射CD34+S1PR1+细胞群移植后饲养及监测一周后即得白血病模型小鼠。
进一步的，移植CD34+S1PR1+细胞后，获得骨髓植入率明显增高，外周血以及肝脾白血病细胞浸润明显增多的免疫缺陷鼠即为白血病模型小鼠。
本发明在第四方面提供了一种筛选用于人急性髓系白血病的治疗剂的方法，其特征在于，所述方法包括以下步骤：包括根据前述方法制备白血病模型小鼠，向小鼠施用测试物质，评估小鼠白血病改善的步骤，从而筛选用于人急性髓系白血病的治疗剂。
本发明的发明人利用RNA-seq对CD34+CD38-的LSC富集的细胞群和non-LSC细胞群(包括CD34+CD38+，CD34-CD38+和CD34-CD38-细胞群)进行基因表达差异分析。结果表明，S1PR1在LSC富集的细胞(CD34+CD38-)中的表达量比non-LSC高。在基因表达存在差异的情况下，利用流式细胞术检测了原代AML肿瘤细胞中S1PR1+和S1PR1-群体中的CD34+CD38-的LSC富集的细胞的占比，进一步验证得出相较S1PR1-细胞群，S1PR1+细胞群中CD34+CD38-的LSC富集的细胞的比例明显增高。因此，S1PR1可作为LSC的表面标记物。
为进一步验证S1PR1对于LSC生物学特性的调控作用，在AML细胞系KG1中，敲低了S1PR1基因并在体外进行了集落形成(colony forming assay，CFU)实验以在体外in vitro实验中评估S1PR1对于AML细胞自我更新能力的影响。结果显示，敲低S1PR1基因后，KG1细胞系的集落形成能力显著下降。除此之外，在分选出AML患者骨髓血中CD34+S1PR1+和CD34+S1PR1-的两群细胞后，利用CFU实验及流式细胞术检测细胞周期的实验，以体外ex vivo方式评估表达S1PR1基因对AML细胞自我更新能力及细胞周期的影响，其结果验证了相较CD34+S1PR1-的细胞群，CD34+S1PR1+细胞所形成的CFU数量显著增多，形态较大，且细胞群中处于分裂期的细胞数量显著增多。可见，S1PR1的表达对于LSC维持自我更新及重建白血病的能力起到关键作用，进一步从反向证明该基因可作为白血病干细胞的标记物，二者之间有较大的关联性。
除此之外，还进一步构建了患者来源的小鼠二次移植模型(patient-derivedxenograft model of secondary transplantation)，分别将CD34+S1PR1+和CD34+S1PR1-的两群细胞以尾静脉注射的方式打入免疫缺陷鼠，以体内研究in vivo的方式检测AML细胞是否表达S1PR1基因对于LSC干性维持能力的影响。结果显示，相较携带CD34+S1PR1-细胞的小鼠，打入CD34+S1PR1+细胞的免疫缺陷鼠在一次移植和二次移植中都出现生存时间显著缩短，骨髓植入率明显增高，外周血以及肝脾白血病细胞浸润明显增多。这一实验同样验证了S1PR1对于维持LSC的自我更新及重建白血病的能力起到至关重要的作用。
本发明提供一种优良的人源化AML模型小鼠，通过使用该小鼠阐明AML的复发机制，并开发适合于AML的治疗剂或治疗方法。
附图说明
图1是AML原代细胞中，S1PR1在LSC和non-LSC中的表达量热图。
图2是S1PR1+和S1PR1-原代AML细胞中CD34+CD38-细胞占比流式图(A)及统计图(B)。
图3是AML细胞系中敲低S1PR1基因后的集落形态图(A)和统计图(B)。
图4是CD34+S1PR1+和CD34+S1PR1-的AML细胞集落形态图(A)及统计图(B)。
图5是CD34+S1PR1+和CD34+S1PR1-的AML细胞周期统计图。
图6是二次移植中，注射CD34+S1PR1+和CD34+S1PR1-的AML细胞的免疫缺陷鼠的生存图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例一基因表达差异分析
在本实施例中，发明人收集了3例AML患者骨髓血，并进行骨髓血单个核细胞提取，分选CD34+CD38-的LSC富集的细胞群和non-LSC细胞群(包括CD34+CD38+，CD34-CD38+和CD34-CD38-细胞群)，并对两群细胞进行RNA测序及差异表达分析。具体步骤如下：
(1)骨髓单个核细胞提取：全血离心(参数为1500rpm，5min)后去除上层血浆；取4mL人白细胞分离液置于15mL离心管内，用PBS稀释血细胞并置于人白细胞分离液上层；离心(参数为1800rpm，18min，关闭刹车)后取中间单个核细胞层并计数。
(2)单个核细胞冻存及复苏：用无血清冻存液冻存患者原代细胞，并转移至液氮保存。待患者确诊AML后，37度水浴锅中复苏，并将细胞转移至37度预热的复苏液(成分为IMDM中添加10％BIT和DNA酶)中。
(3)细胞分选：CD34-PE和CD38-APC与细胞室温避光共孵育15分钟，用PBS洗两次后进行流式分选。分选接收管提前用血清润滑。
(4)分选后离心，提取RNA，提取试剂盒品牌为Qiagen货号为#74104，具体步骤参见厂家说明书。RNA测序在Illumina NovaSeq 6000平台进行，利用Hisat2参照人类基因组GRCh38.87进行比对。用Featurecount计算基因表达量，后用R语言函数DEseq2进行差异基因的分析。结果显示，S1PR1在LSC富集的细胞(CD34+CD38-)中的表达量比non-LSC高(图1)。
(5)原代细胞复苏后，标记抗体如下：CD34-PE，CD38-APC和S1PR1-BV421，抗体和细胞室温避光共孵育15分钟，用PBS洗两次后进行流式细胞术分析。利用S1PR1表达情况圈出阳性群S1PR1+和阴性群S1PR1-，并在两群细胞中分别分析CD34+CD38-细胞所占比例，即利用流式细胞术检测了原代AML肿瘤细胞中S1PR1+和S1PR1-群体中的CD34+CD38-的LSC富集的细胞的占比。结果显示，相较S1PR1-细胞群，S1PR1+细胞群中CD34+CD38-的LSC富集的细胞的比例明显增高(图2)。
实施例二基因敲除验证
本实施例在上述基因表达差异的基础上，在AML细胞系KG1中，敲低了S1PR1基因并在体外做了集落形成实验(colony forming assay，CFU)以在体外in vitro实验中评估S1PR1对于AML自我更新及重建白血病的能力的影响。具体步骤如下：
(1)构建敲低S1PR1基因的AML细胞系：利用干扰RNA技术获得敲低S1PR1基因的KG1细胞系，慢病毒购买自上海吉凯基因。KG1细胞系铺板密度为1*105/mL，加入促转染液80μL后加入病毒液，转染病毒的MOI为50，离心(参数设置为37度，1000g，60min)后置入37度孵箱中培养，15小时后更换为新鲜培养基。3天后进行细胞分选，分选出荧光阳性的细胞。用实时荧光定量qPCR的方法进行S1PR1基因相对表达量的检测并由此确定敲低效率。
(2)集落形成实验：集落形成培养基购买自Stem Cell Technologies，货号为#H04435。细胞重悬于含有2％BIT的IMDM中并计数，接种1000到10000个细胞。取1.1mL含有细胞的干细胞培养基置入35mm的小皿中，铺板2个副孔。培养7-14天后观察。
图3是AML细胞系中敲低S1PR1基因后的集落形态图(A)和统计图(B)，结果显示敲低S1PR1基因后，KG1细胞系所形成的集落形态变小，数量减少，集落形成能力显著下降。
实施例三自我更新能力及细胞周期验证
本实施例分选出AML患者骨髓血中CD34+S1PR1+和CD34+S1PR1-的两群细胞，并利用CFU实验及流式细胞术检测细胞周期的实验，以体外ex vivo方式评估表达S1PR1基因对AML细胞自我更新能力及细胞周期的影响。具体步骤如下：
(1)分选CD34+S1PR1+和CD34+S1PR1-细胞群：单个核细胞提取、冻存及复苏参见实施例一(1)及(2)部分。随后用300μL的PBS重悬细胞并过滤为单细胞悬液，标记抗体为10μL的CD34-PE和5μL的S1PR1-APC，室温避光孵育15分钟后用PBS洗两次。随后进行流式分选。
(2)CD34+S1PR1+和CD34+S1PR1-细胞的集落形成实验步骤参见实施例二(2)部分。
(3)细胞周期的测量：每1*106细胞用1mL含有Hoechst培养液(成分为RPMI1640+2％BIT+10μg/μL的Hoechst)重悬，孵育60分钟。用PBS洗一次后，用100μL含有10μg/μL的Hoechst的PBS重悬细胞。加入5μL的CD34-PE和5μL的S1PR1-APC抗体，室温避光孵育15分钟，PBS洗两次后用300μL的PBS重悬细胞，流式细胞术分析。
图4是CD34+S1PR1+和CD34+S1PR1-的AML细胞集落形态图(A)及统计图(B)。
图5是CD34+S1PR1+和CD34+S1PR1-的AML细胞周期统计图。
结果显示，相较CD34+S1PR1-的细胞群，CD34+S1PR1+的AML细胞所形成的CFU数量显著增多，形态较大(图4)。相较CD34+S1PR1-的细胞群，CD34+S1PR1+的细胞群中处于分裂期的细胞数量显著较多(图5)。
实施例四移植模型的制备
本实施例构建了患者来源的小鼠二次移植模型(patient-derived xenograftmodel of secondary transplantation)，分别将CD34+S1PR1+和CD34+S1PR1-的两群细胞以尾静脉注射的方式打入免疫缺陷鼠，以体内研究in vivo的方式检测AML细胞是否表达S1PR1基因对于LSC自我更新及重建白血病能力的影响。
(1)构建患者来源的小鼠移植模型(patient-derived xenograft model，PDX)：6-8周龄的免疫缺陷NPG雌鼠购买自北京维通达生物技术有限公司，进行至少3天的适应性饲养。移植前1天开始喂抗生素水。移植前4小时进行辐照，辐照剂量为1Gy。准备注射的细胞为原代AML细胞中的CD34+S1PR1+和CD34+S1PR1-细胞群，两群细胞通过流式分选获得，具体步骤参见实施例三第(1)部分。于小鼠尾静脉注射白血病细胞。
(2)小鼠一次移植的白血病监测：打入细胞1周后，择期进行小鼠外周血流式监测(标记抗体为hCD45)。记录小鼠生存，小鼠即将死亡时，立即取骨髓并冲出骨髓细胞，骨髓细胞一部分用于流式检测，另一部分冻存。小鼠肝和脾固定，用于后续病理实验。
(3)小鼠二次移植实验：复苏一次移植的小鼠骨髓细胞，利用磁珠分选的方法分选出hCD45+细胞群。免疫缺陷鼠的饲养及辐照参见本例第(1)部分，尾静脉注射白血病细胞至小鼠体内并进行饲养及监测。小鼠二次移植监测方案与一次移植监测方法相同，参见本例第(2)部分。
图6是二次移植中，注射CD34+S1PR1+和CD34+S1PR1-的AML细胞的免疫缺陷鼠的生存图。相较携带CD34+S1PR1-细胞的小鼠，打入CD34+S1PR1+细胞的免疫缺陷鼠在一次移植和二次移植中都出现生存时间显著缩短，白血病细胞骨髓植入率明显增高，外周血以及肝脾白血病细胞浸润明显增多。
综合上述实施例，表明S1PR1可作为具有调控LSC功能的白血病干细胞分子标志物，并能够用于相关疾病的复发率评估，疾病风险评估，预后评估，分层诊断，检测或者辅助检测病程进展情况，以及相关疾病的治疗或相关药物筛选等，还可利用该标记物制备白血病模型小鼠用于科研。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.S1PR1作为白血病干细胞分子标志物的应用，其特征在于，所述标记物能够调控白血病干细胞的自我更新能力。
2.白血病干细胞分子标志物S1PR1在制备用于白血病诊断或治疗预后监测的试剂或试剂盒中的应用。
3.根据权利要求2所述的应用，其特征在于，所述白血病为急性白血病，优选为急性髓系白血病。
4.根据权利要求2或3所述的应用，其特征在于，所述试剂或试剂盒通过检测被试者骨髓白血病干细胞中的S1PR1的表达量，并与正常对照平均值相比较，诊断疾病进展或评价预后。
5.根据权利要求4所述的应用，其特征在于，所述诊断疾病进展或评价预后是包括：所述1)S1PR1在急性髓系白血病患者骨髓血单个核细胞较正常供者来源的骨髓血单个核细胞高表达；且2)S1PR1在急性髓系白血病干细胞较非干性白血病细胞高表达；特别3)S1PR1在在难治和复发后的标本中异常高表达；或4)S1PR1在自我更新及重建白血病能力强的髓系白血病细胞系和耐药细胞系中高表达。
6.S1PR1在制备用于治疗或预防白血病的药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述药物为S1PR1的抑制剂，优选的，所述S1PR1的抑制剂是抑制S1PR1蛋白质活性或蛋白质水平的抑制剂、或者抑制S1PR1的mRNA水平的抑制剂，优选的，所述抑制S1PR1的mRNA水平的抑制剂是其反义核酸序列、siRNA、miRNA、shRNA、dsRNA，或者其它能够抑制S1PR1的mRNA水平的蛋白质、多肽、酶、化合物。
8.一种制备白血病模型小鼠的方法，其特征在于，所述方法包括以下步骤：1)分选出AML患者骨髓血中CD34+S1PR1+细胞，2)小鼠尾静脉注射CD34+S1PR1+细胞群移植后饲养及监测一周后即得白血病模型小鼠。
9.根据权利要求8所述的方法，其特征在于，移植CD34+S1PR1+细胞后，获得骨髓植入率明显增高，外周血以及肝脾白血病细胞浸润明显增多的免疫缺陷鼠即为白血病模型小鼠。
10.一种筛选用于人急性髓系白血病的治疗剂的方法，其特征在于，所述方法包括以下步骤：包括根据权利要求8或9的方法制备白血病模型小鼠，向小鼠施用测试物质，评估小鼠白血病改善，从而筛选用于人急性髓系白血病的治疗剂。
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